The establishment of a polarized epithelium is one of the earliest stages of development and the maintenance of cell polarity is essential throughout development. Even slight failures in the generation or preservation of polarity can cause significant developmental disorders and birth defects. The regulated transport of cargoes to specific sub-cellular domains is essential for both the establishment and maintenance of polarity. Molecular motors of the dynein and kinesin families are necessary for the polarized transport of cargo in epithelial cells, but it is unknown how they select the correct microtubule track to efficiently move to the correct domain. Evidence suggests that modification of the surface of the microtubule filament by post-translational modification of tubulin subunits or decoration by microtubule associated proteins (MAPs) may provide navigational cues for molecular motors. Members of the kinesin superfamily have been shown to preferentially move along microtubules that are marked by tubulin post-translational modifications (PTMs) and the tau/MAP2 family of side-binding MAPs regulates both access of motors to the filament and motility along the filament. We hypothesize that there are subpopulations of microtubules in polarized epithelial cells in which the surface of the filament has been modified by either PTMs or MAP decoration or both, that these microtubules are arranged in the cell to facilitate cargo delivery to specific subcellular domains, and the molecular motors that mediate polarized transport preferentially move along these modified microtubules. Further, we hypothesize that the composition of these microtubule subpopulations may change as cells undergo a developmental transition from 2D polarity to 3D polarity. To test these hypotheses, we will 1. Define the microtubule subpopulations in epithelial cells and determine if the makeup of these subpopulations changes with polarity using a combination of immunocytochemistry, immunoblotting, immunoprecipitation and mass spectrometry. We will use then use RNAi and overexpression studies to determine if MAP decoration depends upon tubulin modification and vice versa. 2. Define the spatial organization of microtubule subpopulations in epithelial cells at different stages of polarity with quantitative image analysis tools. And 3. Test whether molecular motors involved in polarized transport show differential movement on microtubules modified by individual PTMs or combinations of PTMs with in vitro motility assays. PUBLIC HEALTH RELEVANCE: Many cells are structurally complex, with multiple distinct subcellular domains, and to function correctly, these cells must control the delivery of material t each of these domains. Here we seek to understand this process by identifying the tracks for this transport.